Food

Chemistry
Food Chemistry 105 (2007) 619–627
www.elsevier.com/locate/foodchem

Chemical nature, stability and bioefficacies of anthocyanins

from fruit peel of syzygium cumini Skeels
Jyothi M. Veigas a, Mandayam S. Narayan a, Padmere M. Laxman b,
Bhagyalakshmi Neelwarne a,*
a
Plant Cell Biotechnology Department, Central Food Technological Research Institute, Mysore 560020, India
b
Central Instrumentation Facility and Services Department, Central Food Technological Research Institute, Mysore 560020, India

Received 22 November 2006; received in revised form 6 February 2007; accepted 12 April 2007

Abstract

In the present study, anthocyanin pigments from Syzygium cumini fruit peels were characterized and evaluated for their antioxidant
efficacy, and stability as extract and in formulation. Total anthocyanin content was 216 mg/100 ml of extract which is equivalent to
230 mg/100 g fruit on a dry weight basis. Three anthocyanins were identified as glucoglucosides of delphinidin (1), petunidin (2) and
malvidin (3) by HPLC–ESI–MS. The antioxidant capacity of the extract was tested using models, such as DPPH-scavenging, reducing
power assay, lipid peroxidation in rat brain, liver, liver mitochondria, testes and human erythrocyte ghosts. The extract showed 78.2%
DPPH-scavenging at 2.5 ppm, while BHA exhibited only 41.6% activity at the same concentration, thus proving it to be a more efficient
free radical-scavenger than the widely used BHA. One ppm of the extract was equivalent to 3.5 lM ascorbic acid, as estimated by reduc-
ing power assay. Inhibition of rat brain lipid peroxidation was 94.4% at 5.0 ppm concentration. It was almost equally active in all the
biological models, except human erythrocyte ghost cells, where it showed only 48% inhibition at 5.0 ppm. The extract was quite stable at
0 °C with 11% loss in 4 weeks, while the pigment loss in the antitussive formulation was only 13% at 30 °C at the end of 8 weeks. The high
antioxidant activity and relatively high stability of the pigments make S. cumini a potential source of natural colourant as well as
antioxidants.
Ó 2007 Elsevier Ltd. All rights reserved.

Keywords: Anthocyanins; Indian black plum; Java plum; Delphinidin; Electrospray ionization; Malvidin; Mass spectrometry; Petunidin

1. Introduction of use for various medicinal purposes and currently has a

Indian black plum or Java plum is a tropical edible fruit
obtained from the trees of Syzygium cumini Skeels (Syn.
Eugenia jambolana Lam.; Eugenia cumini Druce; Fam;
Myrtaceae). The fruits are oblong berries, deep purple or
bluish in colour with pinkish pulp, having various medici-
nal properties and used in Ayurveda as a stomachic, astrin-
gent, antiscorbutic, diuretic, antidiabetic, and in chronic
diarrhea and enlargement of spleen (Achrekar, Kakliji,
Pote, & Kelkar, 1991; Morton, 1987; Nadkarni, 1954).
The fruit concentrate of S. cumini has a very long history
*
Corresponding author. Tel.: +91 821 2516501; fax: +91 821 2517233.
E-mail address: pcbt@cftri.res.in (B. Neelwarne).
large market for the treatment of chronic diarrhea and
other enteric disorders, including its use as an antimicrobial
(Migliato, 2005). The leaves are found to reduce radiation-
induced DNA damage in cultured human peripheral blood
lymphocytes (Jagetia & Baliga, 2002). Though different
parts of this species are used in herbal formulations, very
few reports are available on the systematic characterization
of chemical components of the fruit.
There have been different reports of the anthocyanin
composition of the fruits. According to one of the earliest
reports, the deep purple colour of the fruit is due to antho-
cyanins, namely delphinidin-3-gentiobioside and malvidin-
3-laminaribioside, along with petunidin-3-gentiobioside
(Venkateswarlu, 1952). Sharma and Sheshadri (1955)

0308-8146/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2007.04.022
620 J.M. Veigas et al. / Food Chemistry 105 (2007) 619–627
reported the presence of cyanidin diglycoside and glyco-
sides of petunidin and malvidin. Another recent report
on S. cumini Lamarck revealed the presence of malvidin-
3-glucoside and petunidin-3-glucoside in the Brazilian vari-
ety (Lago, Gomes, & da Silva, 2004). All these studies
involved preliminary techniques, such as chemical tests,
paper chromatography and UV–visible spectrophotome-
try. HPLC, coupled to ESI–MS, has proved to be a power-
ful tool for the characterization of anthocyanins, involving
minimal sample preparation (Giusti, Rodriguez-Saona,
Griffin, & Wrolstad, 1999), and, with the current availabil-
ity of this technique, proper confirmation of the chemical
nature of each anthocyanin is possible. Therefore, the pres-
ent study aims at the identification of the anthocyanins of
S. cumini by HPLC, coupled with ESI–MS analysis for the
first time.
In addition to their colourful characteristics, anthocya-
nins are known to possess excellent antioxidant properties
(Kong, Chia, Goh, Chia, & Brouillard, 2003). Anthocya-
nins from different sources have been reported to inhibit
lipid peroxidation, and platelet aggregation (Ghiselli, Nar-
dini, Baldi, & Scaccini, 1998), and possess anti-tumor
(Kamei, Hashimoto, Koide, Kojima, & Hasegawa, 1998),
antimutagenic (Yoshimoto et al., 1999), and hepatoprotec-
tive (Obi, Usenu, & Osayande, 1998) properties. Therefore,
the present study was also focussed on evaluating the anti-
oxidative properties of the extract through in vitro chemical
and cellular models. The low stability of anthocyanins at
high temperature and light conditions (Furtado, Figueki-
redo, Chaves das Neves, & Pina, 1993) is a limiting factor
in their application as colorants. Therefore, the present
study assesses the stability of the pigment as an aqueous
extract and in a formulation under different light and tem-
perature conditions.
2. Materials and methods
2.1. Chemicals
Ascorbic acid, a,a-diphenyl-b-picryl hydrazyl (DPPH),
butylated hydroxyl anisole (BHA) and trifluoroacetic
acid (TFA) were procured from Sigma-Aldrich (Stein-
heim, Germany). Acetonitrile and water, used in HPLC
analysis, were of HPLC grade (Merck, Darmstadt, Ger-
many). XAD-7 (Amberlite polymeric adsorbent of 20–
50 mesh) was purchased from Fluka (Germany). Metha-
nol, butanol, acetic acid and trichloro acetic acid (TCA)
were of analytical grade and were purchased from Qual-
igens, India. All other reagents and chemicals were of
analytical grade and purchased from Merck (Darmstadt,
Germany).
2.2. Plant material
The peels of fully ripe berries obtained from the local
market were manually separated and immediately trans-
ferred to solvent for pigment extraction.
2.3. Extraction of anthocyanins
Anthocyanins were extracted with 0.1% HCl in metha-
nol (Francis, 1986) by way of soaking the fruit peel in a
10-fold volume of the solvent for 3 h on an orbital shaker
set at 100 rpm (25 °C ± 1). After filtration, the residue
was repeatedly extracted until the filtrate obtained was
nearly colourless. The extracts were combined and concen-
trated in a Buchi Rotavapor (Flawil, Switzerland) under
vacuum at 30 °C (±1 °C), and partitioned against ethyl
acetate before application onto an Amberlite XAD-7 col-
umn (Andersen, Viksund, & Pedersen, 1995). The column
eluate (henceforth referred to as ‘‘extract”) was concen-
trated to 100 ml and the total anthocyanin content was
determined by the pH differential method (Lee, Durst, &
Wrolstad, 2005).
2.4. Quantification of total phenolics in anthocyanin extract
Since, this extract may contain other phenolics, in
addition to anthocyanins, the total phenolics were deter-
mined, using the method described by Velioglu, Mazza,
Gao, and Oomah (1998). An aliquot (25 ll) of the suit-
ably diluted extract was mixed with 225 ll methanol
and 1.0 ml of aqueous Folin-Ciocalteau reagent (0.2 M)
and allowed to stand at room temperature (27 °C ±
0.5 °C) for 5 min. One ml of 6% w/v sodium carbonate
solution was added to the mixture, followed by incuba-
tion for 90 min at room temperature and the absorbance
was measured at 725 nm, using a UV–visible spectropho-
tometer (Shimadzu 160 A, Japan). A calibration curve of
gallic acid was prepared (ranging from 0.001 to
0.01 mg ml 1). Results were determined from a regression
equation of the calibration curve (y = 0.0945; R2 =
0.9971) and expressed as mg gallic acid equivalents
(GAE) per 100 ml of the extract.
2.5. Paper chromatography
Partially purified anthocyanins were separated on What-
man No. 3 chromatographic paper. Known volumes of the
extract were applied directly to several sheets of paper and
descending chromatography was carried out in butanol:
acetic acid: water (4:1:5). Three distinct, mauve-coloured
bands were eluted with methanol: acetic acid: water
(90:05:05); the eluates collected and concentrated under
reduced pressure in a Buchi Rotavapor at 30 ± 1 °C.
2.6. Analysis of sugars
An aliquot of the partially purified anthocyanin extract
was dissolved in 2 ml of 2 N HCl and heated to 100 °C in a
screw-capped tube for 90 min. Subsequently, the extract
was cooled and extracted with amyl alcohol. The aqueous
layer, containing hydrolysed sugars, was concentrated
under vacuum and chromatographed on Whatman paper
(Francis, 1986) and also analysed by HPLC–MS.
 J.M. Veigas et al. / Food Chemistry 105 (2007) 619–627
2.7. HPLC–MS Analysis
The partially purified samples as well as anthocyanins
separated on paper, were diluted suitably with methanol,
filtered through a 2 lm membrane filter (Millipore, USA)
and analysed by HPLC, using a Waters Alliance 2695
HPLC equipped with an auto sampler and coupled with
a Waters 2696 photodiode array detector and a Q-TOF
UltimaTM mass spectrometer, utilizing the electro spray
ionization (ESI–MS) interface (Waters Corporation, Man-
chester, UK). The chromatographic separation was per-
formed on a Wakosil II C18 reverse phase stainless steel
column, 250  4.6 mm i.d., 5 lm (SGE, Australia) with a
guard column of the same material. The mobile phase
(0.6 ml min 1) consisted of (A) water/acetonitrile (95:05,
v/v) and (B) water/acetonitrile (50:50, v/v) adjusted to
pH 2.5 (with TFA). The gradient was: 0 min, 15%B;
0–20 min, 15–30%B; 20–25 min, 30–35%B; 25–35 min,
35–40%B; 35–42 min, 40%B; 42–43 min, 40–100%B; 43–
48 min, 100B; and 48–49 min, 100–15%B, followed by
5 min for equilibrium at 15%B. Chromatograms were
acquired at 520 nm. Samples (20 ll) were analysed in dupli-
cate. Positive ion spectra of the column eluate were
recorded in the range of m/z 20–2000 at a scan rate of
2 s/cycle under the following conditions: collision energy
10.0; capillary voltage 35 V; cone voltage 100 kV; source
temperature 80 °C; desolvation temperature 150 °C; cone
gas flow 0.4 l/min; desolvation gas flow (8.3 l/min). Argon
was used as the collision gas. Data acquisition and process-
ing was performed using MassLynxTM 4.0 SP4 software
(Micromass).
HPLC–MS of the extract and its ethyl acetate fraction
were carried out in negative ion mode to analyse other
phenolics present in them. Chromatographic separation
was performed on a Phenomenex Gemini 5 lm C18110 A
reverse phase column, 250  4.6 mm i.d. Mobile phase
consisted of (A) 3% aqueous acetic acid and (B) methanol
with a gradient of 100% A for 1 min, 56%B for 24 min and
100% A for the next 2 min at a flow rate of 1.0 ml/min.
Chromatograms were acquired at 280 nm.
2.8. Free radical-scavenging activity
The antioxidant activity of the anthocyanin extract was
measured on the basis of its ability to scavenge the stable
DPPH. Different concentrations (0.5–2.5 ppm) of the
extracts in methanol (2.0 ml) were treated with 0.5 ml of
a 0.5 mM solution of DPPH in methanol. Absorbance at
517 nm was determined after 20 min and the percentage
scavenging activity was calculated against a reagent blank
(Murthy, Singh, & Jayaprakasha, 2002).
2.9. Reducing power assay
The reduction of ferric to ferrous ion by the extracts is
an indication of the potential antioxidant property. The
reducing power of the extracts was determined by the
621
method of Gulcin (2005). Different concentrations of the
extract (0.5–5.0 ppm to the final concentration) in metha-
nol (1.0 ml) were diluted with 2.5 ml of phosphate buffer
(0.2 M; pH 6.6) and mixed with 2.5 ml of 1% aqueous
potassium ferricyanide. After incubation at 50 °C for
20 min, 2.5 ml of 10% trichloroacetic acid were added to
the mixture. An aliquot of the reaction mixture were
diluted with an equal amount of distilled water and absor-
bance was measured at 700 nm after treatment with 0.5 ml
of 0.1% aqueous FeCl3. Increased absorbance of the reac-
tion mixture indicated an increase in reduction capability.
2.10. Anti-lipid peroxidation assays
Lipid peroxidation was assessed (Halliwell & Gutter-
idge, 1989) in rat brain, liver, liver mitochondria, testes
and human erythrocyte ghost cells. For the preparation
of substrate, brain, liver and testes were obtained from nor-
mal Wistar strain rats and washed in ice-cold saline. The
liver was perfused with ice-cold saline before homogeniza-
tion. A 10% w/v homogenate of the tissues was prepared
separately in ice-cold 1.15% KCl using a Teflon Potter–
Elvehjem glass homogeniser. The homogenate was centri-
fuged at 1000 g for 10 min and the supernatant was used
for study. Rat liver mitochondria were prepared according
to the method of Hogeboom (1955). Mitochondrial frac-
tion was finally suspended in 1.15% KCl, so as to contain
approximately 1 mg of protein per ml of suspension.
2.11. Preparation of human erythrocyte ghost
Small amounts of human erythrocytes were prepared
from 10 ml of freshly drawn blood by sedimentation, at
unit gravity, through 4 volumes of 0.75% (w/v) dextran
T-500 at room temperature in Tris-buffered saline (TBS),
pH 7.5 (10 mM Tris-HCl and 150 mM NaCl). The cells
were then washed three times in 10 volumes of TBS supple-
mented with 10 mM glucose, before being resuspended to a
20% (v/v) suspension, with respect to the packed-cell vol-
ume (Kuhlman, 2000).
2.12. Measurement of lipid peroxidation by thiobarbituric
acid (TBA) Assay
Protein estimation in the above three preparations was
done by the method of Lowry, Rosenberg, Farr, and Ran-
dall (1951) and an aliquot containing 500lg protein equiv-
alent was used for lipid peroxidation assay, as described by
Halliwell and Gutteridge (1989). Briefly, aliquots of the tis-
sue homogenates were treated with different concentrations
of the extracts in methanol (0.5 ml) followed by 1.0 ml each
of 10 lM FeSO4 and 0.1 M ascorbic acid and incubated at
37 °C for 60 min. One ml each of 28% TCA and 1% TBA
were added to the reaction mixture and heated for 15 min
at 95 °C. After cooling on ice and centrifuging the samples,
the absorbance of the supernatant was read at 532 nm. The
reaction mixture without the extract served as a control.
622 J.M. Veigas et al. / Food Chemistry 105 (2007) 619–627
2.13. Formulation of salbutamol syrup and determination of
colour stability
Since, anthocyanins are hydrophilic compounds, they
can be easily incorporated into syrups, which is one of
the most accepted form of pharmaceutical formulations.
The high antioxidant property of the pigments may confer
additional prophylactic advantage, along with the thera-
peutic property of the active ingredient in the syrup. Salbu-
tamol syrup (antitussive) was prepared according to
Howard (1996) and its stability was studied for 8 weeks
under light (2000 Lux) and dark conditions at different
temperatures (20, 30 and 45 °C). Similarly, the stability of
the anthocyanin extract in 1% aqueous solution (pH 3.0)
was monitored for 4 weeks at 0, 10, 20, 30 and 45 °C under
dark conditions. For the stability studies, the test samples
(10 ml) were dispensed into screw-capped tubes and stored.
Each tube was used for one spectral measurement only, so
as to minimize the contact with oxygen. The stability was
assessed by measuring the remaining pigment using spec-
trophotometry at an absorption maximum of 520 nm (Sin-
gha, Baugher, Twonsed, & D’souza, 1991).
3. Results and discussion
3.1. Composition
The results of spectrophotometric analyses of the extract
revealed a total phenolic content (inclusive of anthocya-
nins) of 560 mg GAE/100 ml and an anthocyanin content
of 216 mg/100 ml, the latter being equivalent to 230 mg/
100 g fruit on a dry weight basis. Thus, the total anthocy-
anins in S. cumini are equivalent to blue berries and black
currants that contain 230 and 229 mg/100 g, on a dry
weight basis, respectively and are 1.6-times higher than that
of black berries (141 mg/100 g) (Moyer, Hummer, Finn,
Fig. 1. HPLC separation of anthocyanins of S. cumini. (1) delphinidin-diglucoside; (2) petunidin-diglucoside and (3) malvidin-diglucoside. Detection was
done at 520 nm. Other conditions are explained under the Section ‘Materials and methods’.
Frei, & Wrolstad, 2002). The blue and black berries have
recently been regarded as premier nutraceutical commodi-
ties of great commercial value. Since, S. cumini trees are
perennial, with enormous yields of berries, the information
found through the present study appears to partly comple-
ment its application in various nutraceutical products.
The analysis of the extract by paper chromatography
showed three distinct mauve-coloured bands. The acid
hydrolysis of the S. cumini extract, followed by paper chro-
matography and ESI–MS, revealed the unique presence of
glucose, thus indicating that glucose may be the only sugar
involved in the formation of these anthocyanin glycosides.
No acids were found on alkaline hydrolysis of the anthocy-
anins, thus suggesting the non-acylated nature of the
anthocyanins.
HPLC, coupled to MS, has been a powerful tool for the
characterization of anthocyanins from various sources. A
good HPLC separation was achieved by direct injection
of the partially purified extract (Fig. 1). Three major com-
pounds were identified as glucoglucosides of delphinidin
(1), petunidin (2) and malvidin (3), based on previous work
(Venkateswarlu, 1952) and also supported by their respec-
tive MS. The major anthocyanins, corresponding to peak
1, 2 and 3, represented about 23%, 35% and 38%, respec-
tively of the total peak area revealed at 520 nm. ESI–MS
of each peak resulted in clear and characteristic fragmenta-
tion patterns (Table 1). A typical ESI positive MS shows
two ions: the protonated molecular ion [M + H]+ and a
fragment ion [M + H X]+ arising from loss of the sugar
moiety. However, since the anthocyanins have a natural
residual positive charge, one observes a true molecular
ion [M]+ and a fragment ion [M X]+ which is of the
underivatised aglycone. The value of X, based on the differ-
ence between the molecular ion and fragment, gives a clue
to the nature of the sugar molecule (Abdel-Aal, Young, &
Rabalski, 2006). The compound 1 produced ions at m/z
 J.M. Veigas et al. / Food Chemistry 105 (2007) 619–627 623

Table 1
Assignment of M+ ions for the anthocyanins and their fragments
Compound Retention time (min) ESI(+)–MS/MS m/z
M+ [M+–Glu]+ [M+–Glu–Glu]+
Delphinidin-diglucoside 11.2 627 (100) 465 (40) 303 (100)
Petunidin-diglucoside 15.6 641 (90) 479 (50) 317 (100)
Malvidin-diglucoside 19.8 655 (90) 493 (50) 331 (100)
Values in parentheses represent the relative abundance of the ions.

627, m/z 465 and m/z 303. Similarly, compound 2 showed Fig. 2a–c). This suggests that the aglycones are delphinidin
ions at m/z 641, m/z 479 and m/z 317 and compound 3 pro- (m/z 303), petunidin (m/z 317) and malvidin (m/z 331) for
duced ions at m/z 627, m/z 493 and m/z 331 (Table 1 and compounds 1, 2 and 3, respectively and the differences of
m/z 162 and m/z 321 from the aglycone in the two frag-
ments (in all three compounds) suggest the presence of 2
303.13 627.32 hexoses. The mass spectrum of these compounds revealed
a 100 fragments resulting from the sequential loss of two glucose
627.40
[M+ 162 Da] molecules. However, since both the sugar
303.18 units are glucose (confirmed by paper chromatography of
the acid hydrolysate in comparison with the authentic stan-
dards), it is not possible to determine the sequence of their
%

elimination (Oliveira, Esperanca, & Almoster Ferreira,

465.23 2001).
465.29 Upon paper chromatography of the anthocyanin
extract, only three anthocyanin pigments of intense mauve
628.33
colour were observed. When the methanolic solutions of
0
200 400 600 800 1000 individual spots were analysed by ESI–MS, only the same
m/z three anthocyanins were observed as were identified in
the partially purified total extract.
317.14
b 100 The HPLC pattern of the phenolic components of the
641.33
317.20 anthocyanin extract and the ethyl acetate fraction are
641.42
shown in Fig. 3a and b. The ethyl acetate fraction of the
anthocyanin extract exhibited more peaks with higher
479.24 intensity than did the anthocyanin extract. This suggests
that most of the other phenolics were extracted into ethyl
%

acetate fraction during the partial purification of anthocy-

642.35 anin extract before its application onto the Amberlite
XAD-7 column. In addition, from the X-axis values of
both the chromatograms (Fig. 3a and b; analysed under
0 identical conditions), it could be inferred that the phenolic
200 400 600 800 1000
m/z content in the anthocyanin extract was less than 8% of that
in the ethyl acetate fraction. Therefore we suggest that
c 100 331.17 activity of the extract might be mainly due to the anthocy-
331.23 655.36 anins and the other phenolics play only a minor role in con-
655.44 tributing to the antioxidant activity.

493.27 3.2. Antioxidant activity

493.33
%

DPPH-scavenging assay is useful for rapid analysis of

656.44
free radical-quenching efficacies of various plant extracts
(Murthy et al., 2002). DPPH, a stable free radical with
332.17
an unpaired electron, shows a strong absorption band at
0 517 nm, its solution appearing deep violet in colour. As
200 400 600 800 1000 the electron becomes paired off, which happens in the pres-
m/z
ence of an antioxidant (electron/hydrogen donor), the
Fig. 2. ESI–MS spectra of anthocyanins of S. cumini. (a) delphinidin- absorption vanishes (Blois, 1958). Thus, the faster the reac-
diglucoside; (b) petunidin-diglucoside and (c) malvidin-diglucoside. tion, the more potent is the free radical-scavenging ability
624 J.M. Veigas et al. / Food Chemistry 105 (2007) 619–627

19.30
1.5e-1
1.4e-1
1.3e-1
1.2e-1
1.1e-1
1.0e-1
9.0e-2
AU

8.0e-2
7.0e-2
6.0e-2
5.0e-2
4.0e-2 9.30
3.0e-2
3.38 18.43 19.93
2.0e-2 5.20
1.0e-2
0.0 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
Time

20.42

3.8
3.6
3.4
3.2
3.0
2.8
2.6
AU

2.4
2.2
2.0
1.8
1.6 22.27
1.4
1.2
1.0 18.63
8.0e-1 9.10 21.68
6.0e-1 15.4316.68 19.40 24.17 25.40
4.0e-1
2.0e-1
0.0 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
Time

Fig. 3. LC/MS chromatograms of (a) anthocyanin extract and (b) ethyl acetate fraction of anthocyanin extract. Chromatograms were recorded at 280 nm.

Table 2
Antioxidant activity of anthocyanin-rich extract of S. cumini fruit peela
Conc. (ppm) % DPPH-scavenging activity Reducing powerb % Protection against lipid peroxidation
Brain Liver Testes LMc EGd
0.5 27.0 ± 1.71 2.16 ± 0.72 68.3 ± 0.22 26.1 ± 1.55 25.8 ± 1.65 25.5 ± 1.57 5.86 ± 0.02
1 46.8 ± 0.72 3.50 ± 0.21 71.2 ± 2.31 57.4 ± 1.08 43.2 ± 0.33 44.1 ± 0.85 32.4 ± 0.65
1.5 62.4 ± 1.02 5.86 ± 0.72 73.6 ± 1.25 62.6 ± 1.23 50.1 ± 0.98 67.1 ± 1.23 35.2 ± 1.68
2.0 71.4 ± 0.85 8.05 ± 1.05 74.7 ± 1.54 67.2 ± 0.98 56.7 ± 1.65 70.5 ± 1.66 38.8 ± 2.03
2.5 78.2 ± 1.03 9.72 ± 0.72 86.7 ± 1.80 70.0 ± 1.55 63.4 ± 2.02 72.8 ± 2.56 40.8 ± 1.65
5.0 – – 94.5 ± 0.11 83.3 ± 1.33 72.3 ± 2.65 86.3 ± 2.05 48.6 ± 1.23
a
Data are expressed as mean ± SD (n = 3).
b
Reducing power is expressed as lM ascorbic acid equivalents.
c
Liver mitochondria.
d
Erythrocyte ghost cells.

(Amarowics, Pegg, Rahimi-Moghaddam, Barl, & Weil,
2004). In the present study, the pigment extract quenched
the DPPH in a dose-dependent manner. At 2.5 ppm, the
activity of the extract was 78.2%, while BHA exhibited only
41.6% activity (data not shown) at the same concentration
(Table 2), thus proving the extract to be a potent free rad-
ical-scavenger than the widely used BHA. A recent study
has reported a 50% DPPH-scavenging with 168 lg/ml of
the hot aqueous infusion prepared from fruit peels of S.
cumini dried for 7 days (Banerjee, Dasgupta, & De,
2005). However, our study establishes that the fresh fruit
peel extract offers the same effect at concentrations as low
as 1.39 lg/ml suggesting that the drying and heating pro-
cess might lead to the loss of thermolabile bioactive sub-
stances e.g. anthocyanins and other polyphenols.
The reduction capacity of the extracts is directly propor-
tional to the green/blue colour produced, due to the reduc-
tion of Fe3+/ferricyanide complex to the ferrous (Fe2+)
form. The Fe2+ can be monitored by measuring the forma-
tion of Perl’s Prussian blue at 700 nm (Gulcin, 2005). The
Fe3+–Fe2+ transformation, in the presence of the extracts,
increased with increase in concentration of the extracts.
 J.M. Veigas et al. / Food Chemistry 105 (2007) 619–627 625

One ppm of the extract was equivalent to 3.5 lM of ascorbic
acid in bringing about Fe3+–Fe2+ transformation (Table 2).
This again proves the superiority of the anthocyanin-rich
extract of S. cumini over the traditional antioxidants.
The introduction of phenolic compounds into the lipid-
rich bio-membranes effectively inhibits the lipid oxidation
caused by the chain-propagating lipid peroxyl radicals
and ferrous generating enzymatic systems present on them
(Pazos, Lois, Torres, & Medina, 2006). Anthocyanins from
various sources, being polyphenolic in nature, are known
to cause similar effects against various oxidative reactions
(Kong et al., 2003; Cooke, Steward, Gescher, & Marczylo,
2005). In the present study, the S. cumini extract was tested
for its ability to inhibit the iron-induced lipid peroxidation
in rat brain, liver, liver mitochondria, testes and human
erythrocyte ghost cells. The data (Table 2) indicated that
the pigment extract was most effective (94%) against brain
peel was stored for 6 months (Banerjee et al., 2005). The
lower efficacy in the latter study is probably due to the loss
of anthocyanins during heat processing. Through the pres-
ent study, it has been clearly established that the fresh peel
extract of S. cumini is a powerful antioxidant that can find
various food applications.
Stability of a coloured compound is very important in
retaining the appearance of the product in which it is incor-
porated and hence the customer acceptance, as well as the
bio-efficacy. Naturally occurring colorants, such as antho-
cyanins from black carrot, grape, red cabbage, are now fre-
quently used in foodstuffs and pharmaceuticals due to their
a 50
40

% Color loss
lipid peroxidation at 5.0 ppm compared with rat liver
30
(83%), mitochondria (86%) and testes (72%), erythrocyte
ghost cells (48%) being least responsive to anthocyanin 20
treatment. This could possibly be due to the involvement
of a different mechanism in erythrocyte membranes, which 10
may act as barriers to large molecules. From the data, it
can also be observed that the EC50 of the extract was less 0
than 0.5 ppm for brain homogenate while it was more than 7 14 21 28 35 42 49 56
5.0 ppm for erythrocytes. The homogenates of liver and Time (Days)
liver mitochondria were almost equally protected against Light Dark
peroxidation by the anthocyanin extract, indicating a pos-
sible similarity in the mechanism involved (Table 2). BHA b 50
showed approximately 80% activity against all the systems 40
% Color loss

(data not shown), which is very close to that of the antho-

cyanins. The extent of protective effect exerted by the 30
anthocyanins in different tissues is in the following order: 20
rat brain > liver mitochondria > liver > testes > human
erythrocyte ghost cells. In another study, a 400-fold higher 10
quantity (222 lg/ml) of the hot water extract of dried peel 0
of black plum was required to cause a similar effect, and 7 14 21 28 35 42 49 56
further decrease of activity occurred when the dried fruit Time (Days)
Light Dark
50
c 50
40
% Color Loss

40
% Color loss

30
30
20
20
10
10
0
0
0 10 20 30
7 14 21 28 35 42 49 56
Time (Days)
Time (Days)
0 °C 10 °C 20 ° C Light Dark
30 °C 45 ° C
Fig. 5. Loss of colour in the salbutamol formulation when stored at (a)
Fig. 4. Stability of anthocyanin-rich extract of S. cumini fruit peel. 20 °C; (b) 30 °C and (c) 45 °C under light (2000 Lux) and dark conditions.
Aqueous solution (1%) of the extract (pH 3.0) were stored in screw-capped The formulation was stored in screw-capped vials. Samples from two of
vials. Samples from two of the vials were taken afresh every week and the vials were taken afresh every week and absorbance measured at
absorbance measured at 520 nm. 520 nm.
626 J.M. Veigas et al. / Food Chemistry 105 (2007) 619–627
additional health benefits (Markakis, 1982; Teresa, Buelga,
& Gonzalo, 2002). However, anthocyanins are relatively
unstable, especially when non-acylated (Kirca, Ozkan, &
Cemeroglu, 2007) and are prone to degradation in the pres-
ence of light, high temperature and varying pH (Cemero-
glu, Velioglu, & Isik, 1994; Fossen, Cabrita, & Anderson,
1998). Therefore, the present study aimed at determining
the colour stability in the concentrated extract, as well as
in a pharmaceutical preparation containing salbutamol, a
widely accepted antitussive. Thermal stability of the pig-
ment extract stored in the dark was studied at 0, 10, 20,
30 and 45 °C for 4 weeks and the syrup containing antho-
cyanins extract was studied at 20, 30 and 45 °C in the dark
and in the presence of a continuous illumination of 2000
Lux for 8 weeks. The colour loss in both the extract and
the syrup increased with increase in temperature/time and
more so in presence of light in the case of syrup. At the
end of 4 weeks, the colour losses in the extract were 11.8
and 19.6% at 0 °C and 10 °C, respectively (Fig. 4). The
syrup exhibited a 13% loss when stored at 20 °C and
30 °C in the dark (Fig. 5a and b) after 8 weeks. The pres-
ence of light, along with high temperature, accelerated
the loss of colour in the syrup (Fig. 5c). However, there
was no observable difference in the colour of the extract/
formulation to the naked eye. Since, the syrups are gener-
ally stored in amber-coloured containers, the pigment from
S. cumini appears to be useful for similar formulations that
are stored on the shelf at room temperature. However, the
extract is more stable when stored at 0 °C.
4. Conclusions
The present work reports the chemical nature of antho-
cyanins present in the berries of S. cumini by HPLC–MS
for the first time. The high antioxidant activity of the extract
at extremely low concentrations makes S. cumini a potential
source of antioxidants, as well as a natural colourant. The
S. cumini anthocyanins, like all the other anthocyanins,
have the advantage of high solubility in aqueous mixtures,
imparting an attractive colour that makes easy their incor-
poration into numerous aqueous food and non-food formu-
lations, including pharmaceuticals. S. cumini extract is a
rich source of anthocyanins whose content is equivalent
to that of blue berries and black currants and higher than
that of blackberries, all widely acclaimed anthocyanin-rich
edible fruits (Singha et al., 1991). Like grape anthocyanins,
that are sold commercially as oenocyanin, the peel powder
of S. cumini may also be employed as a colorant for foods
and pharmaceuticals.
Acknowledgement
Authors are grateful to Dr. V. Prakash, Director,
CFTRI, for his encouragement of the work. JMV is grate-
ful to the Council for Scientific and Industrial Research,
Govt., of India, for the grant of a fellowship.
References
Abdel-Aal, E. M., Young, J. C., & Rabalski, I. (2006). Anthocyanin
composition in black, blue, pink, purple, and red cereal grains. Journal
of Agricultural and Food Chemistry, 54, 4696–4704.
Achrekar, S., Kakliji, G. S., Pote, M. S., & Kelkar, S. M. (1991).
Hypoglycemic activity of Eugenia jambolana and Ficus bengalensis:
mechanism of action. In vivo, 5(2), 143–147.
Amarowics, R., Pegg, R. B., Rahimi-Moghaddam, P., Barl, B., & Weil, J.
A. (2004). Free-radical scavenging capacity and antioxidant activity of
selected plant species from the Canadian Prairies. Food Chemistry, 84,
551–562.
Andersen, O. M., Viksund, R. I., & Pedersen, A. T. (1995). Malvidin 3-(6-
acetylglucoside)-5-glucoside and other anthocyanins from flowers of
Geranium sylvaticum. Phytochemistry, 38, 1513–1517.
Banerjee, A., Dasgupta, N., & De, B. (2005). In vitro study of antioxidant
activity of Syzygium cumini fruit. Food Chemistry, 90, 727–733.
Blois, M. S. (1958). Antioxidant determination by the use of a stable free
radical. Nature, 181, 1199–1200.
Cemeroglu, B., Velioglu, S., & Isik, S. (1994). Degradation kinetics of
anthocyanins in sour cherry juice and concentrate. Journal of Food
Science, 59, 1216–1218.
Cooke, D., Steward, W. P., Gescher, A. J., & Marczylo, T. (2005).
Anthocyans from fruits and vegetables – Does bright colour signal
cancer chemo preventive activity?. European Journal of Cancer 41,
1931–1940.
Fossen, T., Cabrita, P., & Anderson, O. M. (1998). Colour and stability of
pure anthocyanins influenced by pH including the alkaline region.
Food Chemistry, 63, 435–440.
Francis, F. J. (1986). Analysis of anthocyanins. In P. Markakis (Ed.),
Anthocyanins as food colors (pp. 181–207). New York, USA: Academic
press.
Furtado, P., Figuekiredo, P., Chaves das Neves, H., & Pina, F. (1993).
Photochemical and thermal degradation of anthrocyanidins. Journal of
Photochemistry and Photobiology, A, 75, 113–118.
Ghiselli, A., Nardini, M., Baldi, A., & Scaccini, C. (1998). Antioxidant
activity of different phenolics fractions separated from Italian red wine.
Journal of Agricultural and Food Chemistry, 46(2), 361–367.
Giusti, M. M., Rodriguez-Saona, L. E., Griffin, D., & Wrolstad, R. E.
(1999). Electrospray and tandem mass spectroscopy as tools for
anthocyanin characterization. Journal of Agricultural and Food Chem-
istry, 47, 4657–4664.
Gulcin, I. (2005). Antioxidant activity of caffeic acid (3, 4-dihydroxycin-
namic acid). Toxicology, 217, 213–220.
Halliwell, B., & Gutteridge, J. M. C. (1989). Free radicals in biology and
medicine (second ed.). Japan: Japan Scientific Societies Press.
Hogeboom, G. H. (1955). In S. P. Colowick & N. O. Kaplan (Eds.).
Methods Enzymology (Vol. I, pp. 16–19). New York: Academic Press.
Howard, C. A. (1996). Introduction to pharmaceutical dosage forms.
Philadelphia: Lea and Febiger.
Jagetia, G. C., & Baliga, M. S. (2002). Syzygium cumini (Jamun) reduces
the radiation-induced DNA damage in the cultured human peripheral
blood lymphocytes: A preliminary study. Toxicology Letters, 132,
19–25.
Kamei, H., Hashimoto, Y., Koide, T., Kojima, T., & Hasegawa, M.
(1998). Antitumor effect of methanol extracts from red and white
wines. Cancer Biotherapy and Radiopharmacology, 13(6), 447–452.
Kirca, A., Ozkan, M., & Cemeroglu, B. (2007). Effects of temperature,
solid content and pH on the stability of black carrot anthocyanins.
Food Chemistry, 101, 212–218.
Kong, J., Chia, L., Goh, N., Chia, T., & Brouillard, R. (2003). Analysis
and biological activities of anthocyanins. Phytochemistry, 64, 923–933.
Kuhlman, P. A. (2000). Characterization of the actin filament capping
state in human erythrocyte ghost and cytoskeletal preparations.
Biochemical Journal, 349, 105–111.
Lago, E. S., Gomes, E., & da Silva, R. (2004). Extraction and anthocyanic
pigment quantification of the Jamun fruit (Syzygium cumini Lamark).
 J.M. Veigas et al. / Food Chemistry 105 (2007) 619–627
http://www.iqsc.usp.br/outros/eventos/2004/bmcfb/trabalhos/docs/
trab-76.pdf.
Lee, J., Durst, R. W., & Wrolstad, R. E. (2005). Determination of total
monomeric anthocyanin pigment content of fruit juices, beverages,
natural colorants and wines by the pH differential method: Collabo-
rative study. Journal of AOAC International, 88, 1269–1278.
Lowry, O. H., Rosenberg, N. J., Farr, A. L., & Randall, R. J. (1951).
Protein measurement with the Folin-phenol reagent. Journal of
Biological Chemistry, 193, 265–267.
Markakis, P. (1982). Anthocyanins as food additives. In P. Markakis
(Ed.), Anthocyanins as food colors (pp. 245–253). United Kingdom:
Academic Press.
Migliato, K. F. (2005). Standardization of the extract of. Syzygium cumini
(l.) skeels fruits. through the antimicrobial activity. Caderno de
Farmácia, 21(1), 55–56.
Morton, J. (1987). Fruits of warm climates. Miami, FL. p. 375.
Moyer, R. A., Hummer, K. E., Finn, C. E., Frei, B., & Wrolstad, R. E.
(2002). Anthocyanins, phenolics and antioxidant capacity in diverse
small fruits: Vaccinium, rubus and ribes. Journal of Agricultural and
Food Chemistry, 50, 519–525.
Murthy, K. N. C., Singh, R. P., & Jayaprakasha, G. K. (2002).
Antioxidant activities of Grape (Vitis vinifera) pomace extracts.
Journal of Agricultural and Food Chemistry, 50, 5909–5914.
Nadkarni, K. M. (1954). In Indian Materia Medica (Vol. 1). India:
Dhootapapeshwar Prakashan Ltd., p. 516.
Obi, F., Usenu, I., & Osayande, J. (1998). Prevention of carbon tetrachloride
induced hepatotoxicity in rat by Hibiscus rosasinensis anthocyanins
extract administered in ethanol. Toxicology, 131(2–3), 93–98.
627
Oliveira, M. C., Esperanca, P., & Almoster Ferreira, M. A. (2001).
Characterisation of anthocyanins by electrospray inonisaiton. Rapid
Communications in Mass Spectrometry, 15, 1525.
Pazos, M., Lois, S., Torres, J. L., & Medina, I. (2006). Inhibition of
Hemoglobin- and Iron-Promoted Oxidation in Fish Microsomes by
Natural Phenolics. Journal of Agricultural and Food Chemistry, 54,
4417–4423.
Sharma, J. N., & Sheshadri, T. R. (1955). Survey of anthocyanins from
Indian sources: Part II. Journal of Scientific and Industrial Research,
14, 211–214.
Singha, S., Baugher, T. A., Twonsed, E. C., & D’souza, M. C. (1991).
Anthocyanin distribution in delicious apples and the relationship
between anthocyanin concentration and chromaticity values. Journal
of American Society for Horticulture Science, 116, 497–499.
Teresa, S. P., Buelga, C. S., & Gonzalo, J. C. R. (2002). LC–MS analysis
of anthocyanins from purple corn cob. Journal of Agricultural and
Food Chemistry, 82, 1003–1006.
Velioglu, Y. S., Mazza, G., Gao, L., & Oomah, B. D. (1998). Antioxidant
activity and total phenolics in selected fruits, vegetables and
grain products. Journal of Agricultural and Food Chemistry, 46,
4113–4117.
Venkateswarlu, G. (1952). On the nature of the colouring matter of the
jambul fruit (Eugenia jambolana). Journal of Indian Chemical Society,
29(6), 434–437.
Yoshimoto, M., Okuno, S., Yoshinaga, M., Yamakawa, O., Yamaguchi,
M., & Yamada, J. (1999). Antimutagenicity of sweet potato (Ipomoea
batatas) roots. Bioscience, Biotechnology and Biochemistry, 63(3),
537–541.